Antibody for human translational regulator

ABSTRACT

The invention provides a human translational regulator (TRANAC) and polynucleotides which identify and encode TRANAC. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of TRANAC.

This application is a divisional application of U.S. application Ser.No. 09/215,063, filed on Dec. 17, 1998, now U.S. Pat. No. 6,365,714,issued Apr. 2, 2002, which is a divisional application of U.S.application Ser. No. 08/869,733, filed Jun. 5, 1997, now U.S. Pat. No.5,955,278, issued Jun. 21, 1999, all of which are entitled “NewTranslational Regulator,” the disclosures of which are incorporatedherein by reference.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of a newhuman translational regulator and to the use of these sequences in thediagnosis, prevention, and treatment of inflammation and disordersassociated with cell proliferation and apoptosis.

BACKGROUND OF THE INVENTION

Protein synthesis is an indispensable process by which living organismsgrow, differentiate, and propagate. The three stages of proteinsynthesis are initiation, elongation, and termination. In eukaryotes,the first event in protein synthesis is the attachment of a freemolecule of methionine (Met) to the end of a tRNA. Met-tRNA and a smallribosomal RNA subunit 40S bind to the mRNA near the AUG initiation codonto form the 40S complex. Addition of the 60S ribosomal RNA subunit tothe 40S complex forms the peptidyl-tRNA transfer site. Once the mRNA,40S, and 60S complexes are in position, peptide synthesis may begin.

Translation initiation factors, eIF1a, eIF2, and eIF3, initiate theformation of the 40S complex and are part of the complex as well.Binding between the 40S ribosomal RNA subunit and the mRNA is aided byeIF4a, eIF4b, and eIF4f using energy from the hydrolysis of GTP bound toeIF2. Translation initiation factor eIF5 promotes the hydrolysis ofribosome-bound GTP producing the energy necessary to bind the 40S and60S complexes.

eIF4f is a complex which recognizes 5′-mGpppN, the CAP structure of alleukaryotic mRNAs. The complex facilitates the association of the 40Ssubunit to mRNA for translation initiation. Translation initiation isregulated by the phosphorylation of the CAP-binding protein, eIF4e, asubunit of the eIF4f complex. Addition of insulin to adipocytes ormuscle cells increases phosphorylation of eIF4e and stimulatesinitiation of protein synthesis (Morley, S. J. & Traugh, J. A. (1990) J.Biol. Chem. 265: 10611-10616). Overexpression of eIF4e has beenassociated with cell transformation (Lararis-Karatzas, A. et al. (1990)Nature 345: 544-547).

PHAS-I, PHAS-II and 4E-BP1 are three regulators of translationinitiation (Pause, A. et al. (1994) Nature 371: 762-767; Hu, C. et al.(1994) Proc. Natl. Acad. Sci. 91: 3730-3734; and Lin, T. -A. andLawrence, J. C. Jr. (1996) J. Biol. Chem 271: 30199-30204). Theassociation of PHAS-I or 4E-BP1 with eIF4e prevents the formation of theactive CAP-binding complex, eIF4f. Phosphorylation of PHAS-I or 4E-BP1by an insulin- or a growth factor-dependent kinase releases eIF4e from acomplex with PHAS-I or 4E-BP1 and prepares eIF4e to bind CAP fortranslation initiation. PHAS-I and PHAS-II are found to have overlappingbut different patterns of expression in tissues. Phosphorylation of bothPHAS proteins promotes dissociation of PHAS-eIF4e complexes andstimulates cell growth (Lin et al., supra).

The discovery of a new human translational regulator and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention and treatmentof inflammation and disorders associated with cell proliferation andapoptosis.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, humantranslational activator (TRANAC), having the amino acid sequence shownin SEQ ID NO:1, or fragments thereof.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or fragments thereof. In a particularaspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2 orvariants thereof.

In addition, the invention provides a polynucleotide sequence whichhybridizes under stringent conditions to the polynucleotide sequence ofSEQ ID NO:2. In another aspect the invention provides a compositioncomprising an isolated and purified polynucleotide sequence encodingTRANAC.

The invention further provides a polynucleotide sequence comprising thecomplement of the polynucleotide sequence encoding the amino acidsequence of SEQ ID NO:1, or fragments or variants thereof. In aparticular aspect, the polynucleotide sequence is the complement of SEQID NO:2. In another aspect the invention provides a compositioncomprising an isolated and purified polynucleotide sequence comprisingthe complement of SEQ ID NO:2, or fragments or variants thereof.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding TRANAC under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified TRANAC having the amino acid sequence of SEQ IDNO:1 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist which decreases theactivity of a polypeptide of SEQ ID NO:1. In one aspect, the inventionprovides a purified antibody which binds to a polypeptide comprising atleast a fragment of the amino acid sequence of SEQ ID NO:1.

Still further, the invention provides a purified agonist which modulatesthe activity of the polypeptide of SEQ ID NO:1.

The invention also features a method for treating or preventinginflammation by administering TRANAC, a method for treating orpreventing cancer by administering TRANAC, a method for treating orpreventing an disorder associated with apoptosis by administering anantagonist of TRANAC, and a method for stimulating cell proliferation byadministering an antagonist of TRANAC.

The invention also provides a method for detecting a polynucleotidewhich encodes TRANAC in a biological sample comprising the steps of: a)hybridizing a polynucleotide sequence complementary to TRANAC (SEQ IDNO:1) to nucleic acid material of a biological sample, thereby forming ahybridization complex; and b) detecting the hybridization complex,wherein the presence of the complex correlates with the presence of apolynucleotide encoding TRANAC in the biological sample. In a preferredembodiment, prior to hybridization, the nucleic acid material of thebiological sample is amplified by the polymerase chain reaction.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO:1) and nucleicacid sequence (SEQ ID NO:2) of TRANAC. The alignment was produced usingMACDNASIS PRO software (Hitachi Software Engineering Co. Ltd. San Bruno,Calif.).

FIG. 2 shows the amino acid sequence alignments among TRANAC (SEQ IDNO:1) and a human translational regulator, 4E-BP1 (GI 561632; SEQ IDNO:3), and a mouse translational regulator, PHAS-II (GI 1658516; SEQ IDNO:4), produced using the multisequence alignment program of DNASTAR™software (DNASTAR Inc, Madison Wis.).

FIGS. 3A and 3B show the hydrophobicity plots for TRANAC, SEQ ID NO:1and 4E-BP1 (SEQ ID NO:3), respectively. The positive X axis reflectsamino acid position, and the negative Y axis, hydrophobicity (MACDNASISPRO software).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

TRANAC, as used herein, refers to the amino acid sequences ofsubstantially purified TRANAC obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

The term “agonist”, as used herein, refers to a molecule which, whenbound to TRANAC, increases or prolongs the duration of the effect ofTRANAC. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of TRANAC.

An “allele” or “allelic sequence”, as used herein, is an alternativeform of the gene encoding TRANAC. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding TRANAC as used herein includethose with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent TRANAC. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding TRANAC,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding TRANAC. The encoded protein may also be “altered” and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent TRANAC.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of TRANAC is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

“Amino acid sequence” as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragment thereof, and to naturallyoccurring or synthetic molecules. Fragments of TRANAC are preferablyabout 5 to about 15 amino acids in length and retain the biologicalactivity or the immunological activity of TRANAC. Where “amino acidsequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

“Amplification” as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term “antagonist” as used herein, refers to a molecule which, whenbound to TRANAC, decreases the amount or the duration of the effect ofthe biological or immunological activity of TRANAC. Antagonists mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich and decrease the effect of TRANAC.

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fa, F(ab′)₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind TRANACpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

The term “biologically active”, as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic TRANAC, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms “complementary” or “complementarity”, as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

A “composition comprising a given polynucleotide sequence” as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding TRANAC (SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2and fragments thereof) may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

“Consensus”, as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, has been extended usingXL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′ directionand resequenced, or has been assembled from the overlapping sequences ofmore than one Incyte Clone using a computer program for fragmentassembly (e.g., GELVIEW™ Fragment Assembly system, GCG, Madison, Wis.).Some sequences have been both extended and assembled to produce theconsensus sequence.

The term “correlates with expression of a polynucleotide”, as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding TRANAC in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

A “deletion”, as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to TRANAC orthe encoded TRANAC. Such modifications include, for example, replacementof hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Human artificial chromosomes (HACs) are linear microchromosomes whichmay contain DNA sequences of 10K to 10M in size and contain all of theelements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The term “hybridization”, as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term “hybridization complex”, as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An “insertion” or “addition”, as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

“Microarray” refers to a high-density array of distinct polynucleotidesor oligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

The term “modulate”, as used herein, refers to a change in the activityof TRANAC. For example, modulation may cause an increase or a decreasein protein activity, binding characteristics, or any other biological,functional or immunological properties of TRANAC.

“Nucleic acid sequence” as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. “Fragments” are thosenucleic acid sequences which are greater than 60 nucleotides than inlength, and most preferably includes fragments that are at least 100nucleotides or at least 1000 nucleotides, and at least 10,000nucleotides in length.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or hybridization assays. As used herein,oligonucleotide is substantially equivalent to the terms “amplimers”,“primers”, “oligomers”, and “probes”, as commonly defined in the art.

“Peptide nucleic acid”, PNA as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast five nucleotides in length linked to a peptide backbone of aminoacid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

The term “portion”, as used herein, with regard to a protein (as in “aportion of a given protein”) refers to fragments of that protein. Thefragments may range in size from five amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein “comprising atleast a portion of the amino acid sequence of SEQ ID NO:1” encompassesthe full-length TRANAC and fragments thereof.

The term “sample”, as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding TRANAC,or fragments thereof, or TRANAC itself may comprise a bodily fluid,extract from a cell, chromosome, organelle, or membrane isolated from acell, a cell, genomic DNA, RNA, or cDNA(in solution or bound to a solidsupport, a tissue, a tissue print, and the like.

The terms “specific binding” or “specifically binding”, as used herein,refers to that interaction between a protein or peptide and an agonist,an antibody and an antagonist. The interaction is dependent upon thepresence of a particular structure (i.e., the antigenic determinant orepitope) of the protein recognized by the binding molecule. For example,if an antibody is specific for epitope “A”, the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled “A” and the antibody will reduce the amount of labeled A boundto the antibody.

The terms “stringent conditions” or “stringency”, as used herein, referto the conditions for hybridization as defined by the nucleic acid,salt, and temperature. These conditions are well known in the art andmay be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

The term “substantially purified”, as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

A “substitution”, as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

A “variant” of TRANAC, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have “nonconservative” changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The Invention

The invention is based on the discovery of a new human translationalregulator (hereinafter referred to as “TRANAC”), the polynucleotidesencoding TRANAC, and the use of these compositions for the diagnosis,prevention, or treatment of inflammation and disorders associated withcell proliferation and apoptosis.

Nucleic acids encoding the TRANAC of the present invention were firstidentified in Incyte Clone 805296 from brain stem tissue cDNA library(BSTMNOT01) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones 882099(THYRNOT02), 805296 (BSTMNOT01), 636579 (NEUTGMT01), and 1254309(LUNGFET03).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A and 1B.TRANAC is 100 amino acids in length and has high abundance of proline,serine, threonine, and glycine. TRANAC also has two potential caseinkinase II phosphorylation sites encompassing residues S76-E79 andT86-E89. As shown in FIG. 2, TRANAC has chemical and structural homologywith 4E-BP1 (GI 561632; SEQ ID NO:3) and PHAS-II (GI 1658516; SEQ IDNO:4). In particular, TRANAC shares 60% and 57% identity with 4E-BP1 andPHAS-II, respectively. As illustrated by FIGS. 3A and 3B, TRANAC and4E-BP1 have rather similar hydrophobicity plots. Northern analysis showsthe expression of this sequence in various libraries, at least 42% ofwhich are immortalized or cancerous, at least 12% of which involveimmune response, and at least 15% of which involve infant/fetal tissuesor organs.

The invention also encompasses TRANAC variants. A preferred TRANACvariant is one having at least 80%, and more preferably 90%, amino acidsequence identity to the TRANAC amino acid sequence (SEQ ID NO:1). Amost preferred TRANAC variant is one having at least 95% amino acidsequence identity to SEQ ID NO:1.

The invention also encompasses polynucleotides which encode TRANAC.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of TRANAC can be used to produce recombinant molecules whichexpress TRANAC. In a particular embodiment, the invention encompassesthe polynucleotide comprising the nucleic acid sequence of SEQ ID NO:2as shown in FIGS. 1A-C.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding TRANAC, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring TRANAC, and all such variations are tobe considered as being specifically disclosed.

Although nucleotide sequences which encode TRANAC and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring TRANAC under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding TRANAC or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding TRANAC and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode TRANAC and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding TRANAC orany fragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency as taught in Wahi, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification Systemmarketed by Gibco/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; M J Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

The nucleic acid sequences encoding TRANAC may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions. When screening for full-length cDNAs, it ispreferable to use libraries that have been size-selected to includelarger cDNAs. Also, random-primed libraries are preferable, in that theywill contain more sequences which contain the 5′ regions of genes. Useof a randomly primed library may be especially preferable for situationsin which an oligo d(T) library does not yield a full-length cDNA.Genomic libraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devisecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. Genotyper™ and Sequence Navigator™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode TRANAC may be used in recombinant DNAmolecules to direct expression of TRANAC, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressTRANAC.

As will be understood by those of skill in the art, it may beadvantageous to produce TRANAC-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter TRANACencoding sequences for a variety of reasons, including but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding TRANAC may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of TRANAC activity, it may be useful toencode a chimeric TRANAC protein that can be recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between the TRANAC encoding sequenceand the heterologous protein sequence, so that TRANAC may be cleaved andpurified away from the heterologous moiety.

In another embodiment, sequences encoding TRANAC may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of TRANAC, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of TRANAC, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active TRANAC, the nucleotidesequences encoding TRANAC or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding TRANAC andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding TRANAC. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding TRANAC,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for TRANAC. For example, when largequantities of TRANAC are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding TRANAC may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding TRANAC may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196.

An insect system may also be used to express TRANAC. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding TRANAC may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of TRANAC will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which TRANAC may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding TRANAC may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing TRANAC in infected host cells (Logan, J. andShenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding TRANAC. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding TRANAC, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressTRANAC may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding TRANAC isinserted within a marker gene sequence, transformed cells containingsequences encoding TRANAC can be identified by the absence of markergene function. Alternatively, a marker gene can be placed in tandem witha sequence encoding TRANAC under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding TRANAC and express TRANAC may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding TRANAC can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding TRANAC. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding TRANAC to detect transformantscontaining DNA or RNA encoding TRANAC.

A variety of protocols for detecting and measuring the expression ofTRANAC, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson TRANAC is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding TRANAC includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding TRANAC, orany fragments thereof may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Minn.);Promega (Madison, Wis.); and U.S. Biochemical Corp., Cleveland, Ohio).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding TRANAC may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeTRANAC may be designed to contain signal sequences which directsecretion of TRANAC through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding TRANAC tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and TRANAC may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingTRANAC and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying TRANAC from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of TRANAC may beproduced by direct peptide synthesis using solid-phase techniquesMerrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using an Applied Biosystems 431Apeptide synthesizer (Perkin Elmer). Various fragments of TRANAC may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

Therapeutics

Chemical and structural homology exists between TRANAC and twotranslational regulators, 4E-BP1 (GI 561632) and PHAS-II (GI 1658516).Northern analysis shows that the expression of TRANAC is associated withcell proliferation, fetal and infant development, inflammation, andimmune response.

Decreased expression of TRANAC appears to be associated with increasedcell proliferation. Therefore, in one embodiment, TRANAC or a fragmentor derivative thereof may be administered to a subject to prevent ortreat a disorder associated with excessive cell proliferation. Suchdisorders include various types of cancer including, but not limited to,adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma, and particularly, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus.

In another embodiment, an agonist of TRANAC or a derivative or fragmentthereof may be used to modulate the activity of TRANAC and to prevent ortreat a disorder associated with excessive cell proliferation including,but not limited to, those listed above.

In still another embodiment, a vector capable of expressing TRANAC, or afragment or a derivative thereof, may be used to prevent or treat adisorder associated with excessive cell proliferation including, but notlimited to, those listed above.

In another embodiment, TRANAC or a fragment or derivative thereof may beadministered to a subject to prevent or treat inflammation associatedwith any disorder of immune response including, but are not limited to,Addison's disease, adult respiratory distress syndrome, allergies,anemia, asthma, atherosclerosis, bronchitis, cholecystitus, Crohn'sdisease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma.

In another embodiment, an agonist of TRANAC or a fragment or derivativethereof may be used to modulate the activity of TRANAC and to prevent ortreat inflammation associated with any disorder including, but notlimited to, those listed above.

In still another embodiment, a vector capable of expressing TRANAC, or afragment or a derivative thereof, may be used to prevent or treatinflammation associated with any disorder including, but not limited to,those listed above.

Increased expression of TRANAC results in apoptosis in normal fetaldevelopment. However, increased expression of TRANAC in other subjectsmay result in apoptosis which may have detrimental effects. Therefore,in one embodiment, an antagonist of TRANAC or a fragment or derivativethereof may be administered to a subject to prevent or treat a disorderwith associated apoptosis. Such disorders include, but are not limitedto, AIDS and other infectious or genetic immunodeficiencies,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, retinitis pigmentosa, andcerebellar degeneration, myelodysplastic syndromes such as aplasticanemia, ischemic injuries such as myocardial infarction, stroke, andreperfusion injury, toxin-induced diseases such as alcohol-induced liverdamage, cirrhosis, and lathyrism, wasting diseases such as cachexia,viral infections such as by hepatitis B and C, and osteoporosis. In oneaspect, an antibody specific for TRANAC may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express TRANAC.

In another embodiment, a vector expressing the antisense orcomplementary sequence of the polynucleotide encoding TRANAC, or afragment or a derivative thereof, may be used to prevent or treat adisorder associated with increased apoptosis including, but not limitedto, those listed above.

In a further embodiment, an antagonist or an inhibitor of TRANAC, or afragment or a derivative thereof, may be added to cells to stimulatecell proliferation. In particular, an antagonist of TRANAC may be addedto a cell or cells in vivo using delivery mechanisms such as liposomes,viral based vectors, or electroinjection for the purpose of promotingregeneration or differentiation of the cell or cells. In addition, anantagonist of TRANAC may be added to a cell, cell line, tissue or organculture in vitro or ex vivo to stimulate cell proliferation for use inheterologous or autologous transplantation. In some cases, the cell willhave been selected for its ability to fight an infection or a cancer orto correct a genetic defect such as sickle cell anemia, β thalassemia,cystic fibrosis, or Huntington's chorea. In one aspect, an antibodyspecific for TRANAC may be used directly as an antagonist, or indirectlyas a targeting or delivery mechanism for bringing a pharmaceutical agentto cells or tissue which express TRANAC.

In a still further embodiment, a vector expressing the antisense orcomplementary sequence of the polynucleotide encoding TRANAC, or afragment or a derivative thereof, may be used to stimulate cellproliferation, as detailed above.

In other embodiments, any of the therapeutic proteins, antagonists,antibodies, agonists, complementary sequences or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

Antagonists or inhibitors of TRANAC may be produced using methods whichare generally known in the art. In particular, purified TRANAC may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind TRANAC.

Antibodies to TRANAC may be generated using methods that are well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith TRANAC or any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to TRANAC have an amino acid sequence consisting of atleast five amino acids and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of TRANAC amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Monoclonal antibodies to TRANAC may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S.L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceTRANAC-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for TRANAC mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between TRANAC and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering TRANAC epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingTRANAC, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding TRANAC may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding TRANAC. Thus, complementary molecules orfragments may be used to modulate TRANAC activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding TRANAC.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encodingTRANAC. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

Genes encoding TRANAC can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes TRANAC. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′ or regulatory regions of the gene encodingTRANAC (signal sequence, promoters, enhancers, and introns).Oligonucleotides derived from the transcription initiation site, e.g.,between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding TRANAC.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding TRANAC. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections orpolycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of TRANAC, antibodies toTRANAC, mimetics, agonists, antagonists, or inhibitors of TRANAC. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of TRANAC, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example TRANAC or fragments thereof, antibodies ofTRANAC, agonists, antagonists or inhibitors of TRANAC, which amelioratesthe symptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind TRANAC may beused for the diagnosis of conditions or diseases characterized byexpression of TRANAC, or in assays to monitor patients being treatedwith TRANAC, agonists, antagonists or inhibitors. The antibodies usefulfor diagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for TRANAC includemethods which utilize the antibody and a label to detect TRANAC in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringTRANAC are known in the art and provide a basis for diagnosing alteredor abnormal levels of TRANAC expression. Normal or standard values forTRANAC expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to TRANAC under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of TRANACexpressed in subject, control and disease, samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingTRANAC may be used for diagnostic purposes. The polynucleotides whichmay be used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofTRANAC may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofTRANAC, and to monitor regulation of TRANAC levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding TRANAC or closely related molecules, may be used to identifynucleic acid sequences which encode TRANAC. The specificity of theprobe, whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding TRANAC, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe TRANAC encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring TRANAC.

Means for producing specific hybridization probes for DNAs encodingTRANAC include the cloning of nucleic acid sequences encoding TRANAC orTRANAC derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding TRANAC may be used for the diagnosisof conditions, disorders, or diseases which are associated withexpression of TRANAC. Examples of such disorders include: various typesof cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, and teratocarcinoma, and particularly, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; disorders associated withinflammation such as Addison's disease, adult respiratory distresssyndrome, allergies, anemia, asthma, atherosclerosis, bronchitis,cholecystitus, Crohn's disease, ulcerative colitis, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjögren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections and trauma; and disorders associated apoptosis such as AIDSand other infectious or genetic immunodeficiencies, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis, retinitis pigmentosa, and cerebellar degeneration,myelodysplastic syndromes such as aplastic anemia, ischemic injuriessuch as myocardial infarction, stroke, and reperfusion injury,toxin-induced diseases such as alcohol-induced liver damage, cirrhosis,and lathyrism, wasting diseases such as cachexia, viral infections suchas by hepatitis B and C, and osteoporosis. The polynucleotide sequencesencoding TRANAC may be used in Southern or northern analysis, dot blot,or other membrane-based technologies; in PCR technologies; or indipstick, pin, ELISA assays or microarrays utilizing fluids or tissuesfrom patient biopsies to detect altered TRANAC expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding TRANAC may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingTRANAC may be labeled by standard methods, and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the biopsied or extractedsample is significantly altered from that of a comparable controlsample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding TRANAC in the sample indicates thepresence of the associated disease. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or in monitoring the treatment of anindividual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of TRANAC, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes TRANAC, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding TRANAC may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5′->3′) and another with antisense (3′<-5′),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of TRANACinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation.

In further embodiments, oligonucleotides derived from any of thepolynucleotide sequences described herein may be used as probes inmicroarrays. The microarrays can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents.

In one embodiment, the microarray is prepared and used according to themethods described in PCT application WO95/11995 (Chee et al.), Lockhart,D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al.(1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which areincorporated herein in their entirety by reference.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs fixed to a solidsupport. Microarrays may contain oligonucleotides which cover the known5′, or 3′, sequence, or contain sequential oligonucleotides which coverthe full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. The oligomers are synthesized atdesignated areas on a substrate using a light-directed chemical process.The substrate may be paper, nylon or other type of membrane, filter,chip, glass slide or any other suitable solid support.

In another aspect, the oligomers may be synthesized on the surface ofthe substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available devises (slot blot or dot blotapparatus) materials and machines (including robotic instruments) andcontain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144dots, or any other multiple which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using the microarrays, the RNA orDNA from a biological sample is made into hybridization probes. The mRNAis isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

In another embodiment of the invention, the nucleic acid sequences whichencode TRANAC may also be used to generate hybridization probes whichare useful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome or to artificial chromosome constructions, such as humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions orsingle chromosome cDNA libraries as reviewed in Price, C. M. (1993)Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

Fluorescent in situ hybridization (FISH as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in various scientific journals or at Online Mendelian Inheritancein Man (OMIM). Correlation between the location of the gene encodingTRANAC on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, TRANAC, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenTRANAC and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to TRANAC large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with TRANAC, or fragments thereof, and washed. Bound TRANAC isthen detected by methods well known in the art. Purified TRANAC can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding TRANAC specificallycompete with a test compound for binding TRANAC. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with TRANAC.

In additional embodiments, the nucleotide sequences which encode TRANACmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I BSTMNOT01 cDNA Library Construction

The BSTMNOT01 cDNA library was constructed from normal brain stemtissue. The donor was a 72 year old male who had died of a myocardialinfaction (specimen #RA95-05-0323; International Institute for AdvancedMedicine, Exton, Pa.).

Cryopreserved tissue was homogenized and lysed using a BrinkmannPOLYTRON PT-3000 Homogenizer (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with phenol chloroform pH8.0, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water and DNase treated at 37° C. Extractionand precipitation were repeated as before. The mRNA was then isolatedusing the QIAGEN OLIGOTEX mRNA purification kit (QIAGEN, Chatsworth,Calif.) and used to construct the cDNA library.

The mRNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat.#18248-013; Gibco/BRL). cDNAs were fractionated on a Sepharose CL4Bcolumn (Cat. #275105-01; Pharmacia), and those cDNAs exceeding 400 bpwere ligated into pSport I. The plasmid pSport I was subsequentlytransformed into DH5α competent cells (Cat. #18258-012; Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the R.E.A.L.PREP 96 plasmid purification kit (Catalog #26173; QIAGEN). This kitenables the simultaneous purification of 96 samples in a 96-well blockusing multi-channel reagent dispensers. The recommended protocol wasemployed except for the following changes: 1) the bacteria were culturedin 1 ml of sterile Terrific Broth (Catalog #22711, Gibco/BRL) withcarbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, thecultures were incubated for 19 hours after the wells and at the end ofthe incubation, the cells were lysed with 0.3 ml of lysis buffer; and 3)following isopropanol precipitation, the plasmid DNA pellet wasresuspended in 0.1 ml of distilled water. After the last step in theprotocol, samples were transferred to a 96-well block for storage at 4°C.

The cDNAs were sequenced by the method of Sanger F and A R Coulson(1975; J Mol Biol 94:441f), using a Hamilton MICROLAB 2200 liquidtransfer system (Hamilton, Reno, Nev.) in combination with PeltierThermal Cyclers (PTC200 from MJ Research, Watertown, Mass.) and AppliedBiosystems' 377 DNA sequencing systems (Applied Biosystems, Foster City,Calif.); and the reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences of the Sequence Listing as well as the aminoacid sequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases, which contain previously identified and annotated sequences,were searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul, S. F (1993) J.Mol. Evol. 36: 290-300; Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410).

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms such asthe one described in Smith et al. (1992, Protein Engineering 5:35-51),incorporated herein by reference, could have been used when dealing withprimary sequence patterns and secondary structure gap penalties. Thesequences disclosed in this application have lengths of at least 49nucleotides, and no more than 12% uncalled bases (where N is recordedrather than A, C, G, or T).

The BLAST approach, as detailed in Karlin, S. and Altschul, S. F. (1993)Proc. Natl. Acad. Sci. 90: 5873-5877 and incorporated herein byreference, searched for matches between a query sequence and a databasesequence. BLAST evaluated the statistical significance of any matchesfound, and reported only those matches that satisfy the user-selectedthreshold of significance. In this application, threshold was set at10⁻²⁵ for nucleotides and 10⁻¹⁴ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam);and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp) for homology. The relevantdatabase for a particular match were reported as Glxxx±p (where xxx ispri, rod, etc., and if present, p=peptide).

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:

% sequence identity x % maximum BLAST score/100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1-2% error;and at 70, the match will be exact. Homologous molecules are usuallyidentified by selecting those which show product scores between 15 and40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding TRANAC occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of TRANAC Encoding Polynucleotides

The nucleic acid sequence of the Incyte Clone 805296 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension in theantisense direction, and the other was synthesized to extend sequence inthe sense direction. Primers were used to facilitate the extension ofthe known sequence “outward” generating amplicons containing new,unknown nucleotide sequence for the region of interest. The initialprimers were designed from the cDNA using OLIGO 4.06 primer analysissoftware (National Biosciences), or another appropriate program, to beabout 22 to about 30 nucleotides in length, to have a GC content of 50%or more, and to anneal to the target sequence at temperatures of about68° to about 72° C. Any stretch of nucleotides which would result inhairpin structures and primer—primer dimerizations was avoided.

Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. Beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, PCR was performedusing the PTC-200 thermal cycler (PTC200; M.J. Research, Watertown,Mass.) and the following parameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat step 4-6 for 15 additional cyclesStep 8 94° C. for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15min Step 11 Repeat step 8-10 for 12 cycles Step 12 72° C. for 8 min Step13 4° C. (and holding)

A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQUICK PCR purification kit (QIAGEN Inc., Chatsworth,Calif.), and trimmed of overhangs using Klenow enzyme to facilitatereligation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2×Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2×Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for an additional29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (and holding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5′ regulatory sequences using the procedure above, oligonucleotidesdesigned for 5′ extension, and an appropriate genolic library.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 primer analysis software (National Biosciences),labeled by combining 50 pmol of each oligomer and 250 μCi of [γ-³²P]adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPontNEN®, Boston, Mass.). The labeled oligonucleotides are substantiallypurified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn).A aliquot containing 10⁷ counts per minute of the labeled probe is usedin a typical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases (Ase I, Bgl II, Eco RI,Pst I, Xba 1, or Pvu II; DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Microarrays

To produce oligonucleotides for a micro array, the nucleotide sequencedescribed herein is examined using a computer algorithm which starts atthe 3′ end of the nucleotide sequence. The algorithm identifiesoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that would interfere with hybridization. Thealgorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a “gridded” array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

VIII Complementary Polynucleotides

Sequence complementary to the TRANAC-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring TRANAC. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 primer analysis softwareand the coding sequence of TRANAC, SEQ ID NO:1. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the TRANAC-encoding transcript.

IX Expression of TRANAC

Expression of TRANAC is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express TRANAC in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofTRANAC into the bacterial growth media which can be used directly in thefollowing assay for activity.

X Demonstration of TRANAC Activity

TRANAC can be expressed by transforming a mammalian cell line such asCOS7, HeLa or CHO with an eukaryotic expression vector encoding TRANAC.Eukaryotic expression vectors are commercially available, and thetechniques to introduce them into cells are well known to those skilledin the art. The cells are incubated for 48-72 hours after transformationunder conditions appropriate for the cell line to allow expression ofTRANAC. Then, phase microscopy is used to compare the mitotic index oftransformed versus control cells. A decrease in the mitotic indexindicates TRANAC activity.

XI Production of TRANAC Specific Antibodies

TRANAC that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Model 431A peptide synthesizer (PerkinElmer) using fmoc-chemistry, and coupled to keyhole limpet hemocyanin(KLH, Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radio iodinated, goat anti-rabbit IgG.

XII Purification of Naturally Occurring TRANAC Using Specific Antibodies

Naturally occurring or recombinant TRANAC is substantially purified byimmunoaffinity chromatography using antibodies specific for TRANAC. Animmunoaffinity column is constructed by covalently coupling TRANACantibody to an activated chromatographic resin, such as CnBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing TRANAC is passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of TRANAC (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/TRANAC binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and TRANAC iscollected.

XIII Identification of Molecules Which Interact with TRANAC

TRANAC or biologically active fragments thereof are labeled with ¹²⁵IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled TRANAC, washed and any wells withlabeled TRANAC complex are assayed. Data obtained using differentconcentrations of TRANAC are used to calculate values for the number,affinity, and association of TRANAC with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 4(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 100 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear   (vii) IMMEDIATE SOURCE:           (A) LIBRARY: BSTMNOT01          (B) CLONE: 805296     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #1: Met Ser Thr Ser Thr Ser Cys Pro Ile Pro Gl #y Gly Arg Asp Gln Leu 1               5   #                10   #                15Pro Asp Cys Tyr Ser Thr Thr Pro Gly Gly Th #r Leu Tyr Gly Thr Thr            20       #            25       #            30Pro Gly Gly Thr Arg Ile Ile Tyr Asp Arg Ly #s Phe Leu Leu Glu Cys        35           #        40           #        45Lys Asn Ser Pro Ile Ala Arg Thr Thr Pro Cy #s Cys Leu Pro Gln Ile    50               #    55               #    60Pro Gly Val Thr Thr Pro Pro Thr Ala Pro Le #u Ser Lys Leu Glu Glu65                   #70                   #75                   #80Leu Lys Glu Gln Glu Thr Glu Glu Glu Ile Pr #o Asp Asp Ala Gln Phe                85   #                90   #                95Glu Met Asp Ile             100 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 432 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear   (vii) IMMEDIATE SOURCE:           (A) LIBRARY: BSTMNOT01          (B) CLONE: 805296     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #2: CTCCTCGACC TCAACGCCAG GCGGTTACTT TGCTGCTCCT NCCGCTCGCT AT#GTCAACGT     60CCACTAGCTG CCCGATTCCC GGGGGCCGGG ACCAGCTGCC CGACTGCTAC AG#CACCACGC    120CGGGGGGCAC GCTATACGGC ACTACCCCCG GAGGCACCAG GATCATCTAC GA#CCGAAAGT    180TCCTGCTGGA GTGCAAGAAC TCACCCATTG CCCGGACAAC CCCCTGCTGC CT#CCCTCAGA    240TTCCCGGGGT CACAACTCCT CCAACAGCCC CTCTYTCCAA GCTGGAGGAG CT#GAAGGAGC    300AGGAGACAGA GGAAGAGATA CCCGATGACG CACAATTTGA AATGGACATC TA#ATCCAGTG    360CAGATGACCT GGCATGTGGA GTTACAGAGG GATCCCTCAT GCCACTGCTG CC#ACCACCTC    420 TTCCTGGGGC AT               #                  #                   #      432 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 120 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (vii) IMMEDIATE SOURCE:          (A) LIBRARY: GenBank           (B) CLONE: 561632    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:Met Ser Ser Ser Ala Gly Ser Gly His Gln Pr #o Ser Gln Ser Arg Ala 1               5   #                10   #                15Ile Pro Thr Arg Thr Val Ala Ile Ser Asp Al #a Ala Gln Leu Pro His            20       #            25       #            30Asp Tyr Cys Thr Thr Pro Gly Gly Thr Leu Ph #e Ser Thr Thr Pro Gly        35           #        40           #        45Gly Thr Arg Ile Ile Tyr Asp Arg Lys Phe Le #u Leu Asp Arg Arg Asn    50               #    55               #    60Ser Pro Met Ala Gln Thr Pro Pro Cys His Le #u Pro Asn Ile Pro Gly65                   #70                   #75                   #80Val Thr Ser Pro Gly Thr Leu Ile Glu Asp Se #r Lys Val Glu Val Asn                85   #                90   #                95Asn Leu Asn Asn Leu Asn Asn His Asp Arg Ly #s His Ala Val Gly Asp            100       #           105       #           110Asp Ala Gln Phe Glu Met Asp Ile         115           #       120(2) INFORMATION FOR SEQ ID NO: 4:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 120 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear   (vii) IMMEDIATE SOURCE:           (A) LIBRARY: GenBank          (B) CLONE: 1658516     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #4: Met Ser Ala Ser Ala Gly Gly Ser His Gln Pr #o Ser Gln Ser Arg Ala 1               5   #                10   #                15Ile Pro Thr Arg Thr Val Ala Ile Ser Asp Al #a Ala Gln Leu Pro Gln            20       #            25       #            30Asp Tyr Cys Thr Thr Pro Gly Gly Thr Leu Ph #e Ser Thr Thr Pro Gly        35           #        40           #        45Gly Thr Arg Ile Ile Tyr Asp Arg Lys Phe Le #u Leu Asp Arg Arg Asn    50               #    55               #    60Ser Pro Met Ala Gln Thr Pro Pro Cys His Le #u Pro Asn Ile Pro Gly65                   #70                   #75                   #80Val Thr Ser Pro Gly Ala Leu Ile Glu Asp Se #r Lys Val Glu Val Asn                85   #                90   #                95Asn Leu Asn Asn Leu Asn Asn His Asp Arg Ly #s His Ala Val Gly Asp            100       #           105       #           110Glu Ala Gln Phe Glu Met Asp Ile         115           #       120

What is claimed is:
 1. An isolated antibody which specifically binds toa polypeptide selected from the group consisting of: a) a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to the amino acid sequence of SEQ ID NO:1, c) a biologicallyactive fragment of a polypeptide having the amino acid sequence of SEQID NO:1, and d) an immunogenic fragment of a polypeptide having theamino acid sequence of SEQ ID NO:1.
 2. A diagnostic test for a conditionor disease associated with the expression of TRANAC in a biologicalsample, the method comprising: a) combining the biological sample withan antibody of claim 1, under conditions suitable for the antibody tobind the polypeptide and form an antibody:polypeptide complex, and b)detecting the complex, wherein the presence of the complex correlateswith the presence of the polypeptide in the biological sample.
 3. Theantibody of claim 1, wherein the antibody is: a) a chimeric antibody, b)a single chain antibody, c) a Fab fragment, d) a F(ab′)₂ fragment, or e)a humanized antibody.
 4. A composition comprising an antibody of claim 1and an acceptable excipient.
 5. A composition of claim 4, wherein theantibody is labeled.
 6. A method of preparing a polyclonal antibody withthe specificity of the antibody of claim 1, the method comprising: a)immunizing an animal with a polypeptide consisting of the amino acidsequence of SEQ ID NO:1, or an immunogenic fragment thereof, underconditions to elicit an antibody response, b) isolating antibodies fromsaid animal, and c) screening the isolated antibodies with thepolypeptide, thereby identifying a polyclonal antibody whichspecifically binds to a polypeptide comprising the amino acid sequenceof SEQ ID NO:1.
 7. A polyclonal antibody produced by a method of claim6.
 8. A composition comprising the polyclonal antibody of claim 7 and asuitable carrier.
 9. A method of making a monoclonal antibody with thespecificity of the antibody of claim 1, the method comprising: a)immunizing an animal with a polypeptide consisting of the amino acidsequence of SEQ ID NO:1, or an immunogenic fragment thereof, underconditions to elicit an antibody response, b) isolating antibodyproducing cells from the animal, c) fusing the antibody producing cellswith immortalized cells to form monoclonal antibody-producing hybridomacells, d) culturing the hybridoma cells, and e) isolating from theculture a monoclonal antibody which specifically binds to a polypeptidecomprising the amino acid sequence of SEQ ID NO:1.
 10. A monoclonalantibody produced by a method of claim
 9. 11. A composition comprisingthe monoclonal antibody of claim 10 and a suitable carrier.
 12. Theantibody of claim 1, wherein the antibody is produced by screening a Fabexpression library.
 13. The antibody of claim 1, wherein the antibody isproduced by screening a recombinant immunoglobulin library.
 14. A methodof detecting a polypeptide comprising the amino acid sequence of SEQ IDNO:1 in a sample, the method comprising: a) incubating the antibody ofclaim 1 with a sample under conditions to allow specific binding of theantibody and the polypeptide, and b) detecting specific binding, whereinspecific binding indicates the presence of a polypeptide comprising theamino acid sequence of SEQ ID NO:1 in the sample.
 15. A method ofpurifying a polypeptide comprising the amino acid sequence of SEQ IDNO:1 in a sample, the method comprising: a) incubating the antibody ofclaim 1 with a sample under conditions to allow specific binding of theantibody and the polypeptide, and b) separating the antibody from thesample and obtaining the purified polypeptide comprising the amino acidsequence of SEQ ID NO:1.